Fractionation of polyalkylene oxide-conjugated hemoglobin solutions

ABSTRACT

Hemoglobin-containing solutions containing polyalkylene oxide-conjugated hemoglobin having a molecular weight greater than about 85,000 daltons and a degree of substitution of at least five polyalkylene oxide conjugates per hemoglobin molecule are described that are not associated with hemoglobinuria in mammals. A method of simultaneously fractionating and purifying polyalkylene oxide-conjugated hemoglobins is also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 616,129, filed on Nov. 20, 1990and now U.S. Pat. No. 5,234,903,which is a continuation-in-part of U.S. patent application Ser. No.440,553, filed on Nov. 22, 1989 and now abandoned. The disclosures ofboth applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to polyalkylene oxide-conjugatedhemoglobins which substantially avoid causing hemoglobinuria in mammals.The present invention also relates to methods for separating theconjugated hemoglobins by degree of polyalkylene oxide substitutionwhile removing endotoxins and phospholipids.

Advances have occurred in recent years in the development ofhemoglobin-based blood substitutes. Such transfusional fluids serve asalternatives to whole blood or blood fractions for use as oxygencarriers and plasma expanders. The use of whole blood and bloodfractions has grown increasingly disfavored because of the risk ofimmune or non-immune reactions and infections, such as acquiredimmunodeficiency syndrome.

The conjugation of polyethylene glycol (PEG) to hemoglobin reduces itsantigenicity and extends its residence time in circulation. However,gross hemoglobinuria was reported by Iwashita and Ajsaka, Organ-DirectedToxic.: Chem. Indicies Mech., Proc. Symp., (Brown et al., Eds. Pergamon,Oxford, England 1981), 97-101 in exchange-transfused rats receivingPEG-conjugates of hemoglobin monomeric subunits below 40,000 daltons.The PEG-conjugation reaction had resulted in dissociation of thehemoglobin tetramer into monomer subunits.

When conjugates having molecular weights over 50,000 daltons wereinfused, hemoglobinuria was not observed. However, Ajisaka and Iwashita,Biochem. Biophys. Res. Comm., 97(3), 1076-81 (1980) disclosed that thesePEG-conjugates of monomeric hemoglobin subunits had P₅₀ 's between 9.5and 12 mm Hg. Such a high oxygen affinity is inefficient for deliveringoxygen to tissues.

Iwasaki and Iwashita, Artif. Organs 10(5), 411-16 (1986) disclose thepreparation of pyridoxalated PEG-hemoglobin conjugates. The conjugateshave weight average molecular weights of 123,000±18,000 daltons and fourto five PEG conjugates per hemoglobin molecule. However, this materialstill exhibited a 5% excretion rate into the urine over a 24 hour periodwhen infused into rats.

U.S. Pat. No. 4,301,144 discloses various polyalkylene oxide hemoglobinconjugates with polyalkylene oxides having molecular weights betweenabout 300 and about 20,000 daltons. The degree of substitution isbetween about 4 and about 120 polyalkylene oxide conjugates perhemoglobin molecule. The conjugate is disclosed as having a circulatinghalf-life of two to four times longer than unmodified stroma-freehemoglobin.

U.S. Pat. No. 4,412,989 discloses various effector molecule modifiedhemoglobins conjugated to polyalkylene oxides. The polyalkylene oxideshave molecular weights between about 300 and about 20,000 daltons and adegree of substitution between about 1 and about 20 conjugates perhemoglobin. A circulating half-life of four to seven times greater thanstroma-free hemoglobin is reported.

U.S. Pat. No. 4,670,417 reports the unsuitability of thehemoglobin-polyalkylene oxide conjugates of U.S. Pat. No. 4,301,144 and4,412,989 because the hemoglobin also denatures during reaction with thepolyalkylene oxide. The conjugation of various hemoglobins topolyalkylene oxides with carboxyl alkylene ether groups is offered as asolution Four to six polyalkylene oxide conjugates per hemoglobin areformed, depending upon whether a dimeric or trimeric intermolecularlycrosslinked conjugate is formed. The polyalkylene oxides disclosed rangein molecular weight from 300 to 20,000 daltons, and the hemoglobin canbe modified with effector molecules. The conjugation of polyalkyleneoxides to hemoglobin via carboxyl alkylene ether linkages, however, iscommercially impractical.

Without being bound by any particular theory, it is believed that theprior art overlooked the possibility that particular low-molecularweight polyalkylene oxide-hemoglobin conjugates were a cause ofhemoglobinuria. It has now been discovered that hemoglobinuria issubstantially eliminated with polyalkylene oxide-hemoglobin conjugateshaving a molecular weight greater than 85,000 daltons and a degree ofsubstitution of five polyalkylene oxide conjugates or greater,regardless of the linkage used between the polymer and the hemoglobin.The molecular weight feature, in combination with the degree ofsubstitution, provides a polyalkylene-oxide conjugated hemoglobinmolecule that is sterically hindered from renal filtration by shape,mass and/or charge and thus not readily causing hemoglobinuria inmammals.

SUMMARY OF THE INVENTION

Therefore, in accordance with the present invention, there are providedsolutions containing polyalkylene oxide-conjugated hemoglobins having amolecular weight greater than about 85,000 daltons and a degree ofsubstitution of at least five polyalkylene oxide conjugates perhemoglobin molecule. The solution of the present invention are notassociated with hemoglobinuria in mammals.

The present invention also includes a method for fractionating solutionscontaining polyalkylene oxide-hemoglobin conjugates of mixed degrees ofsubstitution. This method takes advantage of the fact that theisoelectric point of a conjugated hemoglobin molecule will vary by thedegree of polyalkylene oxide substitution.

The present method also takes advantage of the fact that polyalkyleneoxide-hemoglobin conjugates of varying degrees of substitution can bebound to a variety of anionic stationary phases. By elution underappropriate conditions of buffer ionic strength and pH, the conjugatescan be resolved into fractions varying only by the degree ofsubstitution.

It has also been unexpectedly discovered that the anionic stationaryphases and conditions suitable for the fractionation of solutions ofmixed polyalkylene oxide-hemoglobin conjugates will also result in thebinding and thus the removal of physiologically unacceptable materialssuch as DNA, endotoxins or phospholipids from the solutions. Therefore,the present invention provides a method for simultaneous fractionationand purification of polyalkylene oxide-hemoglobin (PAO-Hb) conjugates.

The method includes:

contacting the PAO-Hb conjugates in solution with an anion exchangeresin capable of selectively binding PAO-Hb conjugates having amolecular weight of less than about 85,000 daltons and a degree ofsubstitution of less than five polyalkylene oxide conjugates perhemoglobin molecule and physiologically unacceptable materials, so thatfractions of conjugated hemoglobin having molecular weights greater thanabout 85,000 daltons and degrees of substitution greater than fivepolyalkylene oxide conjugates per hemoglobin molecule are not bound bythe resin; and

recovering the fractions of conjugated hemoglobins not bound by theresin.

Preferably, the anion exchange resin used in the carrying out of thesimultaneous fractionation/purification process is coated with aquaternary amine.

The above-described method separates the polyalkylene oxide-hemoglobinconjugates of the present invention from lower molecular weight, lesssubstituted fractions, and also serves as a final purification of theconjugates of any endotoxins or phospholipids.

As a result of the present invention, it is possible to providehemoglobin solutions which substantially avoid the problem ofhemoglobinuria and other toxicities associated with prior artmodalities. Moreover, the PAO-Hb conjugates can be purified andfractionated to precise molecular weight ranges and degree ofsubstitution with a single anion exchange chromatography resin-elutionbuffer combination.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The polyalkylene oxide-hemoglobin (PAO-Hb) conjugates of the presentinvention overcome the hemoglobinuria problems associated with prior artcompositions when administered to mammals. The conjugates are preferablyadministered in physiologically-acceptable solutions. The conjugateshave molecular weights greater than about 85,000 daltons and degrees ofsubstitution of five polyalkylene oxides or greater.

The amount of the PAO-Hb conjugate in the solutions of the presentinvention may vary according to the needs of the artisan. It iscontemplated, however, that in most situations, the hemoglobin solutionscontain from about 1.0 to about 10.0 weight percent of the polyalkyleneoxide-hemoglobin conjugates. More preferred solutions contain from about3.0 to about 7.0 weight percent of the conjugates, with amounts of fromabout 4.0 to about 6.0 weight percent being most preferred.

The average degree of substitution, and consequently the averagemolecular weight, may be determined by trinitrobenzene sulfonic acid(TNBS) assay. This technique is well-known and essentially conventional.The molecular weight is determined by multiplying the average number ofconjugates by the molecular weight of the polyalkylene oxide. This isthen added to the molecular weight of the hemoglobin, approximately64,000 daltons. The molecular weight may also be determined bymulti-light channel laser light scattering techniques, which are alsoessentially conventional.

The molecular weight and degree of substitution should be chosen so thatthe viscosity of the solutions do not exceed physiological bloodviscosity of the mammal administered the solution. For humans, thisviscosity ranges from about 4.5 to about 5.0 cps. at 37° C.

Preferred solutions contain polyalkylene oxide-hemoglobin conjugateswith molecular weights between about 90,000 and about 120,000 daltons,with molecular weights of from about 95,000 to about 110,000 daltonsbeing most preferred. These solutions are also preferably substantiallyfree of polyalkylene oxide-hemoglobin conjugates having molecularweights below the preferred ranges. Preferred solutions are also limitedto conjugates having an average degree of substitution of at least about9 conjugates per hemoglobin molecule. More preferred solutions arelimited to conjugates having an average degree of substitution of atleast about 11 conjugates per hemoglobin molecule.

The preferred solutions are substantially free of polyalkyleneoxide-hemoglobin conjugates having degrees of substitution below thepreferred ranges. For purposes of the present invention, substantiallyfree means that the solutions contain no more than about five weightpercent of the hemoglobin conjugate below the molecular weight anddegree of polyalkylene oxide substitution parameters set forth above,and preferably contain less than about one weight percent of suchspecies. In addition, the solutions of the present invention are alsosubstantially free of PAO-Hb conjugates having an average degree ofsubstitution of greater than 18. While such heavily conjugated specieshave not been associated with hemoglobinuria, they are believed to besuboptimal for the purposes of hemoglobin and/or oxygenation ofmammalian tissues. Such species can be eliminated from the inventivesolutions by controlling the PAO-Hb conjugation reaction and/orseparation techniques within the skill of the art.

The polyalkylene oxides include polyethylene glycol (PEG), polypropyleneglycol and block copolymers thereof. To be suitable for use in thepresent invention, the polyalkylene oxides must be soluble in water atroom temperature. Therefore, the degree of block copolymerization forthe block copolymers should not be so great to render the polymer waterinsoluble at room temperature. Polyalkylene oxides having molecularweights between about 1,000 and about 20,000 daltons are suitable foruse with the present invention. Polyalkylene oxides having molecularweights between about 2,000 and about 10,000 daltons are preferred.

The conjugated hemoglobins can be prepared from hemoglobins from anyappropriate mammalian source, human or non-human, depending upon need.At present, the most commercially viable hemoglobins are human andruminant hemoglobins, particularly bovine hemoglobin. Human hemoglobincan be obtained from whole human blood, either freshly drawn or from theoutdated supply of blood banks. Human hemoglobin can also be obtainedfrom placentas or packed erythrocytes obtained from human blood donorcenters. The hemoglobin can also be produced by recombinant methodsincluding the establishment of transgenic herds or cells. Suchtransgenic animals may express wild type human, variant human or mutatedhuman hemoglobin. Solutions of the present invention may also containmixtures of various conjugated hemoglobins.

Ruminant hemoglobin such as bovine or sheep are also useful. Bovinehemoglobin is obtained, for example, from slaughterhouses. The choice ofanimal source is not critical, but will instead be made on the basis ofcommercial demand. The products of the present invention also haveveterinary end-uses. Therefore, various animal sources are appropriatefor the products and methods of the present invention.

The method by which the hemoglobins have been extracted fromerythrocytes is not critical. The extracted hemoglobin preferably has anendotoxin concentration less than about 9.1 EU/mL as measured by gelclot or kinetic turbidometric Limulus Amebocytic Lysate (LAL) assay. Thephospholipid level is preferably non-detectable as determined by HighPerformance Liquid Chromatography (HPLC) lipid assays. Phospholipidlevels of up to 0.50 mg/mL, however, are acceptable and can be removedin accordance with the methods described herein.

Preferably, the hemoglobin is separated by the method disclosed incopending and commonly owned U.S. patent application Ser. No. 913,138,filed Jul. 14, 1992. The disclosure of this application is herebyincorporated herein by reference thereto. The hemoglobin has alsopreferably been purified of endotoxins and phospholipids by the methodsdisclosed in this patent application.

The conjugate is formed by covalently bonding the hydroxyl terminals ofthe polyalkylene oxide and the free amino groups of the lysine residuesof the hemoglobin. Any art-recognized method for conjugatinghydroxyl-terminated polymers with the free amino groups of proteins orpolypeptides is suitable for use with the present invention. Typically,the terminal hydroxyl groups are first activated. This refers to theconversion of the hydroxyl group to a functional derivative capable ofreacting with the free amino groups.

One example of polyalkylene oxide activation is the cyanuric chlorideactivation of polyalkylene oxides disclosed in commonly owned U.S. Pat.No. 4,179,337 to Davis. The disclosure of this patent is herebyincorporated herein by reference thereto. Another art-recognized methodfor activating polyalkylene oxides forms the correspondingsuccinyl-N-hydroxysuccinimide ester. This well-known procedure isdisclosed in Abuchowski et al., Cancer Biochem. Biophys., 7, 175-86(1984).

In a preferred aspect of the invention, urethane linkages are formedwith the protein amino groups and the activated polyalkylene oxides.Preferably, the urethane linkage is formed as described in commonlyowned U.S. Pat. No. 5,122,614, the disclosure of which is herebyincorporated by reference. This patent discloses the formation ofN-succinimide carbonate derivatives of polyalkylene oxides.

The conjugates of hemoglobin and N-succinimide carbonates ofpolyalkylene glycols can also be prepared as described in copending andcommonly owned parent U.S. patent application Ser. No. 440,553, filedNov. 22, 1989 and parent U.S. patent application Ser. No. 616,129, filedNov. 20, 1990. The disclosures of these applications have beenincorporated by reference. However, the deoxygenation step disclosed inapplication Ser. No. 440,553 and the partial deoxygenation stepdisclosed in application Ser. No. 616,129 are now considered optionaland may be omitted if desired.

Regardless of the conjugation method, the reaction forms a mixture ofpolyalkylene oxide-conjugated hemoglobins of varying degrees ofconjugation. The mixture also includes some residual unconjugatedpolyalkylene oxides and hemoglobin. This mixture is typically insolution in a reaction buffer containing one or more of phosphate,chloride and bicarbonate anions.

The mixture of polyalkylene oxide-conjugated hemoglobin (PAO-Hb)reaction products are preferably fractionated in a buffer solutioncontaining from about 1.0 to about 10.0% PAO-Hb conjugates by weight.Suitable solutions have a pH of from about 8.0 to about 9.0 andpreferably from about 8.7 to about 9.0. The buffers also have anosmolality between about 25 and about 110 milliosmoles/kg. Osmolalityranges of between about 33 to about 100 milliosmoles/kg are preferred,while a range of from about 67 to about 100 milliosmoles/kg isespecially preferred. The solutions preferably contain one or morebuffer salts selected from KCl, NaCl, K₂ HPO₄, KH₂ PO₄, Na₂ HPO₄,NaH₂PO₄, NaHCO₃, NaBO₄, (NH₄)₂ CO₃ and glycine NaOH. Sodium borate buffersare preferred for use in the present invention.

Depending upon the reaction buffer utilized, the solution of conjugatedhemoglobins may first have to undergo a buffer exchange. The bufferexchange provides a solution having the required osmolality forfractionation. However, such exchanges are essentially conventional andmay be performed by, for example, ultrafiltration. Typically, thepolyalkylene oxide-conjugated hemoglobin solution is ultrafilteredacross a low molecular weight cut-off (30,000 to 50,000 dalton)membrane.

The fractionation of the polyalkylene oxide-hemoglobin (PAO-Hb)conjugates is preferably carried out by contacting the PAO-Hb conjugatesin solution with an anion exchange medium which is capable ofselectively binding those conjugates having a molecular weight of lessthan 85,000 daltons and a degree of substitution of less than fivepolyalkylene oxide conjugates per hemoglobin molecule. Thisfractionation is achieved by the fact that the conjugated hemoglobinmolecules of various degrees of substitution will have isoelectricpoints which also vary in a somewhat predictable fashion. For example,the isoelectric point of hemoglobin is determined by the number ofavailable lysine residues available on the surface of the protein. Theselysine residues also serve as the point of attachment of polyalkyleneoxide conjugates. Therefore, as the degree of substitution ofpolyalkylene oxide conjugates to hemoglobin increases, the isoionicpoint decreases, and the ability of the polyalkylene oxide-hemoglobinconjugate to bind to an anion exchange resin weakens.

The use of strongly polar anion exchange resins are especially preferredfor the method of the present invention. For this reason, quaternaryamine coated anion exchange resins are utilized. The quaternary amineresin may be coated onto either a polymeric or silica matrix; however,polymeric matrices are preferred. A number of tetramethylamine, orquaternary methylamine, anion exchange resins are commerciallyavailable, coated onto the support matrices. Included among thecommercially available quaternary anion exchange resins suitable for usewith the present invention are QA TRISACRYL® and QMA-SPHEROSIL®,quaternary amine resins coated onto a polymer matrix, manufactured byIBF of Garenne, France, for Sepracor of Marlborough, Mass.; TMAE 650 M®,a tetramethylamino ethyl resin coated onto a polymer matrix,manufactured by EM-Separators of Gibbstown, N.J.; QAE 550 C®, and SUPERQC®, each a quaternary amine resin coated onto a polymer matrix andmanufactured by Toso Haas of Montgomeryville, Pa. QMA Accell,manufactured by Millipore of Millford, Mass. and PEI resins manufacturedby JT Baker of Phillipsburg, N.J., may also be used.

Conventional liquid chromatography, rather than HPLC is preferablyutilized to fractionate the solutions of mixed polyalkyleneoxide-hemoglobin conjugates. That is, techniques of HPLC are notcritical to the fractionation of mixed solutions of polyalkyleneoxide-conjugated hemoglobin. Conventional liquid chromatography is morereadily adapted to large-scale commercial production techniques thanHPLC. Furthermore, a significant economic advantage is also obtained bya reduction in the cost of equipment, process time and risk of endotoxincontamination.

The chromatography columns should have an axial flow or radial flowdesign and a diameter between about 1.6 cm and about 1000 cm. The columnlength should be between about 5 cm and about 1000 cm. Such columns willtypically hold between about 1 mL and about 785 L of anion exchangechromatography resins. The chromatography equipment, anion exchangeresin, and buffers should be depyrogenated, utilizing standardprocedures.

Typically, the anion exchange resin is packed in the column andequilibrated by conventional means. A buffer having the same pH andosmolality as the conjugated hemoglobin solution is used. The conjugatedhemoglobin solution is then absorbed onto the column at a rate of about0.1 to about 0.5 liters a minute. At the completion of the loading, aflow of an elution buffer is applied to the column to elute fractions ofpolyalkylene oxide-conjugated hemoglobin. The fractions are ofessentially uniform molecular weight and degree of substitution.

The elution method is not critical and will depend upon the needs of theend user. A preferred polyalkylene oxide conjugated hemoglobin fractionis collected having a molecular weight greater than about 85,000 daltonsand a degree of substitution of five or more polyalkylene oxideconjugates. The fraction can be obtained by a single step elutionutilizing an isocratic flow of an elution buffer having a pH and anosmolality within the ranges set forth above. The elution bufferpreferably contains one or more salts selected from KCl, NaCl, K₂ HPO₄,KH₂ PO₄, Na₂ HPO₄, NaH₂ PO₄, NaHCO₃, NaBO₄ and (NH₄)₂ CO₃. The preferredfraction is substantially free of lower molecular weight conjugates andhemoglobins with four or fewer polyalkylene oxide conjugates. The lowermolecular weight, less conjugated species, as well as any unconjugatedhemoglobins can then be backwashed from the column by conventionaltechniques.

Gradient elution and techniques utilizing multiple isocratic steps ofincreasing concentration within the osmolality range can also be used.Gradient and multiple isocratic elution steps of increasingconcentration will result in the sequential elution of fractions ofpolyalkylene oxide-hemoglobin conjugates. The degree of polyalkyleneoxide-conjugation within each fraction will be substantially uniform.However, the degree of polyalkylene oxide conjugation for each fractionwill decrease with elution time.

Techniques of flow-through chromatography can also be employed, in whichthe column is first equilibrated by conventional means with a bufferhaving the same pH and osmolality as the conjugated hemoglobin solution.The conjugated hemoglobin solution is then loaded onto the column at arate of about 0.1 to about 0.5 liters a minute. Hemoglobin conjugateshaving a degree of conjugation associated with hemoglobinuria bind tothe column while conjugates that do not cause hemoglobinuria flowthrough and are immediately collected. The preferred buffer forflow-through chromatography is 20 mM NaHCO₃. The preferredchromatography resin is QAE 550 C®, a quaternary amine resin coated ontoa polymer matrix, manufactured by Toso Haas of Montgomeryville, Pa. Thebasic elution and flow-through chromatography techniques describedherein are essentially conventional and can be applied to the inventiveprocesses with the disclosed buffers and chromatography resins by one ofordinary skill without undue experimentation.

The temperature range for elution is between about 4° C. and about 25°C. Preferably, elution is carried out at a temperature of from about 6°C. to about 150° C. and most preferably at about 8° C. The elution ofthe polyalkylene oxide-hemoglobin fractions is detected by UV absorbanceat 280 nm. Fraction collection may be achieved through simple timeelution profiles.

The preferred hemoglobin fractions can then be pooled to provide asolution in the elution buffer of polyalkylene oxide-hemoglobinconjugates. The conjugates have a molecular weight greater than about5,000 daltons and a degree of substitution of five conjugates orgreater. Typically, the pooled fractions have a concentration betweenabout 1.0 and about 10.0 weight percent of the polyalkyleneoxide-hemoglobin conjugate, actual amounts will vary, however.

The pooled hemoglobin fractions are preferably included with aphysiologically-acceptable carrier such as those well-known to those ofordinary skill in the art. For example, a physiologically-acceptablecarrier may have a pH of about 7.8 and include a phosphate/saline buffersystem containing NaCl (100 mM), KCl (10 mM), Na₂ HPO₄ (3 mM) andNaHCO₀₃ (30 mM). If necessary, the pooled fractions may be transferredto a physiologically-acceptable carrier by buffer exchange techniquessuch as ultrafiltration.

Following the collection of the conjugated hemoglobin fractions, thechromatography column should be washed to remove the materials that havebound to the column. The column can then be re-equillibrated andprepared for another loading of polyalkylene oxide-conjugatedhemoglobins to be fractionated.

The present invention also includes the unexpected discovery that whenthe above-described fractionation methods are carried out,physiologically unacceptable materials can also be simultaneouslyremoved by binding to the anion exchange resins. It has been found, forexample, that commonly present by-products which are physiologicallyunacceptable such as DNA, endotoxins or phospholipids bind to the anionexchange resin. The polyalkylene oxide-conjugated hemoglobins are thusalso purified while the conjugates are fractionated. It is preferred,however, that the PAO-Hb conjugates and solutions containing same berendered substantially free of these impurities before the contactingwith the anion exchange resin.

An example of a preferred process is as follows:

About 24 liters of a 5-6 weight percent solution of bovine hemoglobinconjugated with polyethylene glycol (PEG) in 4 mM pH 9.0±0.1 boratebuffer is loaded onto a 60 cm long, 25 cm diameter liquid chromatographycolumn. The column is packed with 30 L of quaternary amine anionexchange resin such as DEAE-spherodex (by IBF of Garenne, France)equilibrated with the above buffer. The PEG-hemoglobin solution isloaded at a rate of 0.50 L a minute, and once it is completely absorbed,an isocratic flow of the buffer is started to elute the PEG-hemoglobinfrom the column. Elution of a PEG-hemoglobin fraction having a degree ofconjugation between about 6 and about 11 PEG conjugates per hemoglobinmolecule is detected with a UV detector. At this point, collection ofthe effluent is initiated and continued until the effluentPEG-hemoglobin peak has been reduced to five percent of peak amplitude.

The foregoing procedure produces PEG-hemoglobin fractions that, whenpooled, typically have a PEG-hemoglobin concentration within theconcentration range of about 1.0 and about 10.0 weight percent,typically about 2.0 weight percent. The pooled fractions typically havean endotoxin level of less than 0.1 EU/mL as measured by gel clot orkinetic turbidometric LAL assay. The phospholipid level is nondetectableas measured by HPLC liquid assay. The pool of fractions can then bestored for extended periods using standard techniques at temperatures ofabout -20° C. for future use or maintained at temperatures of about2°-8° C. for immediate use.

It is contemplated that the present method can be applied to fractionateother polymer-hemoglobin conjugates to resolve the polymer conjugateinto fractions varying only by the degree of polymer substitution. Whenbuffers of the above-described pH and osmolality are utilized, themethod will also purify such other polymer-hemoglobin conjugates ofphysiologically incompatible materials such as DNA, endotoxins andphospholipids.

This aspect of the present invention provides a versatile process bywhich polyalkylene oxide-conjugated hemoglobins can be fractionated andat the same time, purified of unwanted materials such as DNA, endotoxinsand phospholipids. This permits the isolation of polyalkyleneoxide-hemoglobin conjugates which are substantially free of contaminantsand substantially avoid causing hemoglobinuria in mammals. Solutions ofthe conjugates in a pharmaceutically-acceptable carrier are particularlysuitable for use as hemoglobin-based cell-free transfusional fluids.Such transfusional fluids are acceptable as alternatives to whole bloodor blood fractions for use as oxygen carriers and plasma expanders.

The following non-limiting examples illustrate certain aspects of theinvention. These examples are not meant in any way to restrict theeffective scope of the invention. All parts and percentages are byweight unless otherwise noted, and all temperatures are in degreesCelsius.

EXAMPLES Example 1 PREPARATION OF BOVINE HEMOGLOBIN-POLYETHYLENE GLYCOLCONJUGATE

Frozen bovine hemoglobin (bHb) (12.4%, 25 L) was thawed at 4° C.overnight. The thawed bHb was mixed with 25 L of reaction buffer (0.8 mNaCl and 0.2 M Na₂ HPO₄) to make 50 L of 6.2% bHb solution. The pH wasadjusted to 7.8 by adding 1 M KH₂ PO₄ solution while maintaining thetemperature at 8° C.±0.2° C. The hemoglobin solution was deoxygenatedunder nitrogen gas using a gas permeable membrane to achieve a level of75-80% deoxy-bHb. A 12 molar excess of polyethylene glycol-succinimidylcarbonate (SC-PEG), prepared according to the method of U.S. Pat. No.5,122,614, was added to the deoxy-bHb solution and the reaction mixturewas stirred at 8° C. for 2 hours. When the PEG reaction was completed,30 mM cysteine was added into the reaction solution and the pH wasadjusted to 8.0±0.1 with either 1 M KH₂ PO₄ or NaOH. The reactionsolution was then filtered using a 0.22 micron Durapore filter. Thereaction solution was then buffer-exchanged to 3.3 mM borate buffer, pH9.0±0.2 using ultrafiltration equipment (Centrasette 30K).

Example 2

A 30×60 cm column was packed with 30 L DEAE-spherodex (IBF of Garenne,France) in 3.3 mM borate buffer, pH 9.0±0.1. The column wasdepyrogenated with 0.2 N NaOH and equillibrated with four column volumes(120 L) of depyrogenated 3.3 mM borate buffer, pH 9.0. The capacity ofthe DEAE-spherodex for PEG-bHb was determined to be 40 mg/ml.

24 L of a 5 weight percent solution of the PEG-bHb of Example 1 wasloaded onto the ion exchange column. The balance of the PEG-bHb solutionwas stored at -20° C. The column was washed with three column volumes(90 L) of 3.3 mM borate buffer, pH 9.0±0.1 to remove unreacted PEG.PEG-bHb was then eluted with 70 L 100 mM borate buffer, pH 9.0.

The eluted PEG-bHb was then buffer exchanged by ultrafiltration into aformulation buffer. The formulation buffer was prepared by dissolvingthe following salts into 550 L of distilled water:

    ______________________________________                                        NaCl (100 mM)         3.2142  kg                                              KCl (10 mM)           410.08  g                                               Na.sub.2 HPO.sub.4 (3 mM)                                                                           442.31  g                                               NaHCO.sub.3 (30 mM)   1.386   kg                                              ______________________________________                                    

The pH of the formulation buffer was then adjusted to 7.8±0.1 by adding1 M KH₂ PO₄ (HCl or H₃ PO₄ could also have been used). The bufferexchange was then performed by concentrating the volume of the purifiedPEG-bHb solution collected from the ion exchange column to approximately5±0.1 weight percent PEG-bHb using a 50 K Centrasette (Filtron) primedwith 50 L distilled water and 10 L formulation buffer. Ultrafiltrationwas continued until 550 L formulation buffer (20 fold) was consumed. Thecompleteness of the dialysis was checked and the resulting solution wasthen sterile filtered through a Durapore filter (0.22 micron, Millipore)into 300 mL blood bags and stored at -20° C.

The degree of conjugation of the PEG-bHb was determined to beapproximately 9 by trinitrobenzenesulfonic acid (TNBS) assay, awell-known technique. The solution was free of phospholipids.

Example 3

A PEG-bHb solution was fractionated according to the procedure ofExample 2, except that 67 mM borate buffer was used as the elutingbuffer. The average degree of conjugation of the eluted fraction was 11PEG per hemoglobin molecule, determined by TNBS assay.

Example 4

The PEG-bHb of Example 1 was dialyzed with distilled water to removeexcess salt. The dialyzed PEG-bHb was charged on a DEAE-spherodexcolumn, which had been previously equilibrated with 1 mM Na₂ HPO₄ buffersolution, pH 8.03. The column was successively eluted with 2 mM, 5 mM,10 mM and 50 mM Na₂ HPO₄ buffer solutions, pH 8.03. The collectedfractions were concentrated by centrifugation. The fractions weredetermined to decrease in the degree of conjugation as the concentrationof the elution buffer increased.

Example 5

Several PEG-bHb solutions, prepared as described herein wereadministered to laboratory rats by exchange transfusion (E.T.). In thisexample, solutions containing varying degrees of PAO-Hb conjugation weretransfused to demonstrate the lack of hemoglobinuria in mammalsadministered the inventive solutions. The test results are depicted inthe table below.

                  TABLE                                                           ______________________________________                                              Avg. Degree of                                                                            Concentration                                                                             %    mg Hb/mL                                   Sample                                                                              Conjugation (Wt. %)     E.T. Urine                                      ______________________________________                                        1      8.0        6.1         40   0.02 ± 0.003                            2      8.6        4.2         60   0.04 ± 0.004                            3     12.0        6.2         30   0.00                                       4     12.0        6.2         50   0.00                                       5     12.0        6.2         70   0.00                                       ______________________________________                                    

The animals underwent different levels of exchange transfusion in orderto achieve similar dosage because the samples were of differentconcentration. The solutions produced no detectable renal tubularnecrosis.

Hemoglobinuria experiments designed to investigate the relationshipbetween hemoglobinuria and renal injury have determined byhistopathology that PEG-Hb yielding less than 0.1 mg Hb/mL urine at 50%E.T. causes no detectable renal tubular necrosis, whereas PEG-bHbproducing more than 0.1 mg Hb/mL urine results in mild acute tubularnecrosis. As can be seen from the table above, those samples depictedabove are not associated with renal toxicity or pathologicalhemoglobinuria.

Numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and scope of the invention. All such modifications are intendedto be included within the scope of the following claims.

We claim:
 1. A polyalkylene oxide-conjugated hemoglobin-containingsolution comprising polyalkylene oxide-conjugated hemoglobin in aphysiologically acceptable aqueous buffer solution, said polyalkyleneoxide-conjugated hemoglobin having a molecular weight greater than about85,000 daltons and a degree of substitution of at least fivepolyalkylene oxide conjugates per hemoglobin molecule, wherein saidsolution does not produce hemoglobinuria in mammals.
 2. The solution ofclaim 1, wherein the concentration of said polyalkylene oxide-conjugatedhemoglobin in said solution is from about 1 to about 10 weight percent.3. The solution of claim 2, wherein said concentration of saidpolyalkylene oxide-conjugated hemoglobin in said solution is from about3 to about 7 weight percent.
 4. The solution of claim 3, wherein saidconcentration of said polyalkylene oxide-conjugated hemoglobin in saidsolution is from about 4 to about 6 weight percent.
 5. The solution ofclaim 1, wherein said polyalkylene oxide-conjugated hemoglobin has amolecular weight between about 90,000 and about 120,000 daltons.
 6. Thesolution of claim 5, wherein said polyalkylene oxide-conjugatedhemoglobin has a molecular weight between about 95,000 and about 110,000daltons.
 7. The solution of claim 1, wherein said polyalkyleneoxide-conjugated hemoglobin has an average degree of substitution of atleast about 9 polyalkylene oxide conjugates per hemoglobin molecule. 8.The solution of claim 7, wherein said polyalkylene oxide-conjugatedhemoglobin has an average degree of substitution of at least about 11polyalkylene oxide conjugates per hemoglobin molecule.
 9. The solutionof claim 1, wherein said polyalkylene oxide is selected from the groupconsisting of polyethylene glycol, polypropylene glycol and blockcopolymers thereof.
 10. The solution of claim 1, wherein saidpolyalkylene oxide comprises polyethylene glycol.
 11. The solution ofclaim 1, wherein said polyalkylene oxide has a molecular weight betweenabout 1,000 and about 20,000 daltons.
 12. The solution of claim 11,wherein said polyalkylene oxide has a molecular weight between about2,000 and about 10,000 daltons.
 13. The solution of claim 1, whereinsaid hemoglobin comprises mammalian hemoglobin.
 14. The solution ofclaim 13, wherein said hemoglobin comprises human hemoglobin.
 15. Thesolution of claim 13, wherein said hemoglobin comprises ruminanthemoglobin.
 16. The solution of claim 15, wherein said ruminanthemoglobins comprise bovine hemoglobins.
 17. The solution of claimwherein said hemoglobin comprises a hemoglobin produced by recombinantmethods.
 18. The solution of claim 1, wherein said polyalkylene oxidesare conjugated to free amino groups of the lysine residues of saidhemoglobin.
 19. The solution of claim 18, wherein said polyalkyleneoxides are conjugated to said lysine residues of said hemoglobin bymeans of urethane linkages.
 20. The solution of claim 1, wherein saidsolution is substantially free of lower molecular weight, lessconjugated polyalkylene oxide-conjugated hemoglobin species.
 21. Thesolution of claim 20, wherein said lower molecular weight, lessconjugated species are present at levels of less than about one weightpercent.